1. Field of the Invention
The present invention comprises human DNA compositions encoding proteins that confer potassium channel activity to membranes or recipient cell lines. The DNA compositions include structural genes coding for the potassium channel proteins, expression and replication plasmids or vectors containing the structural genes and host cells expressing those genes. Methods of screening compounds for potassium channel modulating activity are also described.
2. Description of the Related Art
Ho, K., et al. "Cloning and expression of an inwardly rectifying ATP-regulated potassium channel." Nature (4 Mar. 1993) Vol. 362 pp. 31-38. Describes the gene that encodes an ATP-regulated potassium channel protein from the inner stripe of outer medulla of rat kidneys.
Chandy, K., et. al., WO 92/02634, PCT/US91/05168, published 20 Feb. 1992. Describes the gene product known as MK3, a voltage dependent, type n potassium channel protein in T lymphocytes.
Harpold, M., et. al., WO92/02639, PCT/US91/05625, published 20 Feb. 1992. Transcription assays that identify compounds that modulate the activity of cell surface proteins. Cells that contains DNA that encode reporter genes, transcriptional control elements and heterologous cell surface proteins that may be potassium ion channels.
Luzdunski, M., "Potassium Channels: Structure-Function Relationships, Diversity, and Pharmacology," Cardiovascular Drugs and Therapy, (1992) Vol. 6, pp. 312-319. General description and information concerning potassium channels.
3. Background
Ionic channels of cell membranes are the basic sites where ionic fluxes take place. The modem era of the study of drug-channel interactions began when voltage clamp techniques were used to demonstrate the block of Sodium, (Na.sup.+), and potassium, (K.sup.+), channels of squid axons caused by procaine and cocaine. Narahashi, Ann Neurology (1984); 16(suppl): S39-S51.
This invention concerns potassium channels. Pharmacological and biophysical studies have revealed multiple subtypes for membrane ion channels that form potassium selective pores in the plasma membrane of many mammalian cells. Comparison of the pharmacological and electrophysiological properties of these potassium channels has given rise to an operational definition for grouping the various subtypes based largely on their gating properties.
Voltage-gated potassium channels sense changes in membrane potential and pass potassium ions in response to this alteration in the cell membrane potential. Ligand-gated potassium channels are regulated by small molecular weight effectors which include calcium, sodium, ATP or fatty acids (particularly arachidonic acid). Lazdunski, Cardiovascular Drugs and Therapy (1992) Vol. 6 pp. 313-319. Although these channel proteins share the common property that they selectively move potassium ions, their distinct biophysical, biochemical and pharmacological properties suggests that they are different gene products encoded by distinct genes.
The ATP-Sensitive, or ATP-gated, potassium channel is an important class of channels that links the bioenergetic situation of the cell to its electrical excitability. The channel is blocked by high intracellular ATP concentrations and it opens when ATP decreases. Lazdunski (1992). Although ATP-gated potassium channels were originally described in cardiac tissue; Noma, A. Nature (1983) Vol. 305 pp. 147-148, they have subsequently been described in pancreatic .beta.-cells; Cook et. al., Nature (1984) Vol. 311 pp. 271-273, vascular smooth muscle; Nelson, M. T. et. al., Am. J. Physiol. (1990) Vol. 259 pp. C3-C18 and in the thick ascending limb of the kidney; Wang, W. et. al. Am. J. Physiol. (1990) Vol. 258, pp. F244-F-253.
Molecular cloning studies on potassium channel proteins has yielded some information, but only for members of the voltage-gated family of potassium channels. Various genes encoding these voltage-gated family of potassium channel proteins have been cloned using Drosophila genes derived from both the Shaker, Shaw and Shab loci; Wei, A. et. al., Science (1990) Vol. 248 pp. 599-603.
All known attempts to clone members of the ligand-gated family of potassium channel proteins using probes based on the known sequences of voltage-gated potassium channels have been unsuccessful. Taken together with the electrophysiological and pharmacological properties of these potassium channels, these results further confirm that the ATP-gated channel proteins are encoded by genes that are distinct from the genes that encode for voltage-gated potassium channels. These results indicate that there is little or no homology between the genes encoding voltage-sensitive potassium channels and the ATP-gated potassium channels.
A cDNA encoding a rat kidney potassium channel has been isolated by expression cloning, using a size-fractionated mRNA from the rat kidney thick ascending limb. When expressed in oocytes, the protein encoded by this gene displays many but not all of the hallmarks of a ATP-gated potassium channel. Ho, K., et al., Nature, Vol. 362 pp. 31-38 (4 Mar. 1993).
This invention describes the first successfully cloned human kidney ATP-gated and related potassium channel genes. Cloning these important genes will allow the production of important channel proteins into systems that will permit the identification, characterization and cloning of potential drug targets. This invention includes the development of a high volume mechanistic screen and it will allow the production of appropriate systems for the production of material for biochemical study.
The discovery of the highly selective and potent sodium channel blocking action of tetrodotoxin ignited a widespread interest in using specific chemicals as probes for the study of ionic channels. Narahashi (1984). The present invention provides an important potassium channel that may allow the discovery of other important physiological compounds.